Project Summary Multidrug-resistant bacteria are increasingly the cause of infectious disease fatalities worldwide. Because civilization is at the precipice of a post-antibiotic era, alternative methods to treat infectious diseases need to be utilized. One especially insidious bacterial pathogen, Clostridium difficile, thrives in the human gut and releases its toxin after antibiotic treatment diminishes the commensal intestinal microbiota. C. difficile infection (CDI) can cause profuse diarrhea and colitis and is one of the leading causes of gastroenteritis related fatalities. CDI can be difficult to treat, as C. difficile can form dormant spores during treatment with antibiotics and cause relapse after treatment has ended. There is evidence fecal microbiota transplantation or treatments with probiotic bacteria can prevent or treat CDI. However, the mechanistic basis for probiotic-based protection is not fully understood. Recent work from the Hang Lab at Rockefeller University has identified secreted antigen A (SagA), a putative peptidoglycan hydrolase secreted by the commensal bacterium Enterococcus faecium that increased host immunity to CDI in mice. Remarkably, recombinant expression of SagA in other bacterial species also increased mouse tolerance against CDI implicating SagA as a potential therapeutic agent that can be delivered by a probiotic. In order to understand how SagA activity increases host protection, peptidoglycan hydrolase activity of SagA will be characterized. Additionally, stable expression strains of SagA will be engineered in probiotic bacteria. Finally, host protection against CDI will be investigated using the stably engineered strains of probiotic-SagA in a mouse model. The long-term goals of this project are to elucidate the protective mechanism of SagA and engineer probiotics to stably express and secrete SagA for future design of novel therapeutics against enteric bacterial infections. Aim 1: Biochemical characterization of SagA peptidoglycan hydrolase activity Aim 2: Engineer stable expression strains of SagA in probiotic bacteria Aim 3: Investigate protection of SagA-probiotic against Clostridium difficile